Continuous synthesis and anti-myocardial injury of tanshinone IIA derivatives.

A series of tanshinone IIA derivatives were synthesized through sulfonation, slat-forming, chlorination, and amidation reactions. Meanwhile, anti-myocardial injury activity was evaluated in vitro. D8 and D9 exhibited a slightly higher anti-myocardial injury (5.78, 7.46 µM) activity compared with esmolol (8.12 µM). In addition, they also displayed a concentration-dependent inhibition on the anti-myocardial injury.

Clavuridins A and B, two new trinor-guaiane sesquiterpenes isolated from the Xisha soft coral Clavularia viridis.

In the present study, two new trinor-guaiane sesquiterpenes, named clavuridins B (1), and A (2), along with three known sesquiterpenes (3-5), were isolated from the Xisha soft coral Clavularia viridis. Their structures and absolute configurations were determined on the basis of spectroscopic analysis, X-ray diffraction analysis with Cu Kα radiation and by comparison with related model compounds. Compounds 1 and 3-5 were evaluated for their cytotoxic activity.

Discovery of a novel NEDD8 Activating Enzyme Inhibitor with Piperidin-4-amine Scaffold by Structure-Based Virtual Screening.

NEDD8 activating enzyme (NAE) plays an important role in regulating intracellular proteins with key parts in a broad array of cellular functions. Here, we report a structure-based virtual screening of a compound library containing 50 000 small molecular entities against the active site of NAE. Computational docking and scoring followed by biochemical screening and target validation lead to the identification of 1-benzyl-N-(2,4-dichlorophenethyl) piperidin-4-amine (M22) as a selective NAE inhibitor. M22 is reversible for NAE, inhibits multiple cancer cell lines with GI50 values in the low micromolar range, and induces apoptosis in A549 cells. Furthermore, it produces tumor inhibition in AGS xenografts in nude mice and low acute toxicity in a zebrafish model. M22, a novel NAE inhibitor, represents a promising lead structure for the development of new antitumor agents.

Effect of Cholesterol on Cellular Uptake of Cancer Drugs Pirarubicin and Ellipticine.

The cell membrane is a major barrier for drug transport. Given that many cancer drugs must passively cross the cell membrane, understanding drug-membrane interactions is crucial. We used fluorescence-activated cell sorting to investigate how cholesterol influences the transport of the cancer drugs ellipticine and pirarubicin across cell membranes. We showed that cholesterol depletion helped pirarubicin cross the membranes of nonsmall cell lung carcinoma and Chinese hamster ovary cells. In contrast, the uptake of ellipticine was not strongly influenced by cholesterol depletion. To study the microscopic origins of these observations, atomistic molecular dynamics simulations were performed. Doxorubicin (similar in structure to pirarubicin) and ellipticine were simulated in model membranes of POPC and POPC with 40 mol % cholesterol. Atomistic free energy calculations for the translocation of a single ellipticine and doxorubicin across the lipid bilayers qualitatively matched the experiment results. The free energy barrier for doxorubicin crossing the bilayer was strongly increased when cholesterol was present, while for ellipticine the barrier remained similar with and without cholesterol. Molecular dynamics simulations showed that the different hydrogen-bonding propensities of the two drugs are likely the major factor for the different behaviors. The qualitative agreement between cell experiments and atomistic computer simulations illustrates the potential to link observed biological phenomena and single molecule mechanisms of actions. Our results suggest that the traditional understanding of drug permeation and the influence of cholesterol on the small molecule transport is naïve and needs to be re-examined.

Design and Characterization of a Multifunctional pH-Triggered Peptide C8 for Selective Anticancer Activity.

Most drug delivery systems have been developed for efficient delivery to tumor sites via targeting and on-demand strategies, but the carriers rarely execute synergistic therapeutic actions. In this work, C8, a cationic, pH-triggered anticancer peptide, is developed by incorporating histidine-mediated pH-sensitivity, amphipathic helix, and amino acid pairing self-assembly design. We designed C8 to function as a pH-responsive nanostructure whose cytotoxicity can be switched on and off by its self-assembly: Noncytotoxic ß-sheet fibers at high pH with neutral histidines, and positively charged monomers with membrane lytic activity at low pH. The selective activity of C8, tested for three different cancer cell lines and two noncancerous cell lines, is shown. Based on liposome leakage assays and multiscale computer simulations, its physical mechanisms of pore-forming action and selectivity are proposed, which originate from differences in the lipid composition of the cellular membrane and changes in hydrogen bonding. C8 is then investigated for its potential as a drug carrier. C8 forms a nanocomplex with ellipticine, a nonselective model anticancer drug. It selectively targets cancer cells in a pH-responsive manner, demonstrating enhanced efficacy and selectivity. This study provides a novel powerful strategy for the design and development of multifunctional self-assembling peptides for therapeutic and drug delivery applications.

Enhanced bioelectricity generation by improving pyocyanin production and membrane permeability through sophorolipid addition in Pseudomonas aeruginosa-inoculated microbial fuel cells.

Improvement on electron shuttle-mediated extracellular electron transfer (EET) is of great potential to enhance the power output of MFCs. In this study, sophorolipid was added to enhance the performance of Pseudomonas aeruginosa-inoculated MFC by improving the electron shuttle-mediated EET. Upon sophorolipid addition, the current density and power density increased ∼ 1.7 times and ∼ 2.6 times, respectively. In accordance, significant enhancement on pyocyanin production (the electron shuttle) and membrane permeability were observed. Furthermore, the conditions for sophorolipid addition were optimized to achieve maximum pyocyanin production (14.47 ± 0.23 µg/mL), and 4 times higher power output was obtained compared to the control. The results substantiated that enhanced membrane permeability and pyocyanin production by sophorolipid, which promoted the electron shuttle-mediated EET, underlies the improvement of the energy output in the P. aeruginosa-inoculated MFC. It suggested that addition of biosurfactant could be a promising way to enhance the energy generation in MFCs.

Bioavailability comparison of a new form of vilazodone XVII to IV in beagles using liquid chromatography/mass spectrometry.

Vilazodone hydrochloride (CAS 163521-12-8) is polymorphic and has 15 crystal forms, referred to as I-XI and XIII-XVI. In the study, we prepared and performed structural identification of a new crystal form named XVII. To investigate this in vivo, a rapid and sensitive method based on liquid-liquid extraction, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed and validated for the determination of vilazodone hydrochloride in dog plasma. This HPLC-MS/MS method was successfully applied to a bioavailability comparison of two crystal forms of vilazodone hydrochloride (IV and XVII) in six healthy beagles using a single-dose, two-way crossover design. The maximum plasma concentration (C(max)), the time taken to reach C(max), and the area under the concentration-time curve were determined following oral administration of 10 mg vilazodone hydrochloride (IV or XVII) to beagles. These analyses revealed no significant bioavailability differences between vilazodone hydrochloride forms IV and XVII in dogs.

Enhancement of bioelectricity generation by cofactor manipulation in microbial fuel cell.

Microbial fuel cells (MFCs) are promising for harnessing bioenergy from various organic wastes. However, low electricity power output (EPT) is one of the major bottlenecks in the practical application of MFCs. In this study, EPT improvement by cofactor manipulation was explored in the Pseudomonas aeruginosa-inoculated MFCs. By overexpression of nadE (NAD synthetase gene), the availability of the intracellular cofactor pool (NAD(H/(+))) significantly increased, and delivered approximately three times higher power output than the original strain (increased from 10.86 µW/cm(2) to 40.13 µW/cm(2)). The nadE overexpression strain showed about a onefold decrease in charge transfer resistance and higher electrochemical activity than the original strain, which should underlie the power output improvement. Furthermore, cyclic voltammetry, HPLC, and LC-MS analysis showed that the concentration of the electron shuttle (pyocyanin) increased approximately 1.5 fold upon nadE overexpression, which was responsible for the enhanced electrochemical activity. Thus, the results substantiated that the manipulation of intracellular cofactor could be an efficient approach to improve the EPT of MFCs, and implied metabolic engineering is of great potential for EPT improvement.

Enhancement of bioelectricity generation by manipulation of the electron shuttles synthesis pathway in microbial fuel cells.

Microbial fuel cells (MFCs) are promising for generating bioenergy and treating organic waste simultaneously. However, low extracellular electron transfer (EET) efficiency between electrogens and anodes remains one of the major bottlenecks in practical applications of MFCs. In this paper, pyocyanin (PYO) synthesis pathway was manipulated to improve the EET efficiency in Pseudomonas aeruginosa-inoculated MFCs. By overexpression of phzM (methyltransferase encoding gene), the maximum power density of P. aeruginosa-phzM-inoculated MFC was enhanced to 166.68 µW/cm(2), which was four folds of the original strain. In addition, the phzM overexpression strain exhibited an increase of 1.6 folds in PYO production and about a onefold decrease in the total internal resistance than the original strain, which should underlie the enhancement of the EET efficiency and the electricity power output (EPT). On the basis of these results, the manipulation of electron shuttles synthesis pathways could be an efficient approach to improve the EPT of MFCs.

Fungal arachidonic acid-rich oil: research, development and industrialization.

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

Ultrasonic pretreatment and acid hydrolysis of sugarcane bagasse for succinic acid production using Actinobacillus succinogenes.

Immense interest has been devoted to the production of bulk chemicals from lignocellulose biomass. Diluted sulfuric acid treatment is currently one of the main pretreatment methods. However, the low total sugar concentration obtained via such pretreatment limits industrial fermentation systems that use lignocellulosic hydrolysate. Sugarcane bagasse hemicellulose hydrolysate is used as the carbon and nitrogen sources to achieve a green and economical production of succinic acid in this study. Sugarcane bagasse was ultrasonically pretreated for 40 min, with 43.9 g/L total sugar obtained after dilute acid hydrolysis. The total sugar concentration increased by 29.5 %. In a 3-L fermentor, using 30 g/L non-detoxified total sugar as the carbon source, succinic acid production increased to 23.7 g/L with a succinic acid yield of 79.0 % and a productivity of 0.99 g/L/h, and 60 % yeast extract in the medium could be reduced. Compared with the detoxified sugar preparation method, succinic acid production and yield were improved by 20.9 and 20.2 %, respectively.

Succinic acid production by Actinobacillus succinogenes NJ113 using corn steep liquor powder as nitrogen source.

In this study, corn steep liquor powder (CSL) was used as nitrogen source to replace the relatively costly yeast extract typically used for the production of succinic acid with Actinobacillus succinogenes NJ113. Moreover, when heme was added to the fermentation medium and the culture was agitated at a low speed, a maximum succinic acid concentration of 37.9 g/l was obtained from a glucose concentration of 50 g/l, and a productivity of 0.75 g/l/h was achieved. These yields are almost as high as for fermentation with glucose and yeast extract. These results suggest that heme-supplemented CSL may be a suitable alternative nitrogen source for a cost-effective method of producing succinic acid with A. succinogenes NJ113 while consuming less energy than previous methods.

Butanol production from acid hydrolyzed corn fiber with Clostridium beijerinckii mutant.

Sulfuric acid treated corn fiber hydrolysate (SACFH) inhibited cell growth and the production of butanol (4.7±0.2 g/L) by Clostridium beijerinckii IB4 in P2 medium. Optimal medium components were determined using fractional factorial design. NH4HCO3, FeSO4·7H2O and CaCO3 were demonstrated to be significant components in the production of butanol. The Box-Behnken design and a corresponding quadratic model were used to predict medium components (NH4HCO3 1.96 g/L, FeSO4·7H2O 0.26 g/L and CaCO3 3.15 g/L) and butanol yield (9.5 g/L). The confirmation experiment, under the predicted optimal conditions, yielded a butanol level of 9.5±0.1g/L. This study indicates that the Box-Behnken design is an effective approach for screening the optimal medium components required for the production of butanol. It also demonstrates that SACFH, which has high levels of inhibitors such as furan and phenolic compounds, may be used as a renewable carbon source in the production of biofuels.

An oxidoreduction potential shift control strategy for high purity propionic acid production by Propionibacterium freudenreichii CCTCC M207015 with glycerol as sole carbon source.

The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0-160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L(-1), productivity 0.192 g L(-1) h(-1), the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD(+), ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source.

Butanol production from hemicellulosic hydrolysate of corn fiber by a Clostridium beijerinckii mutant with high inhibitor-tolerance.

A Clostridium beijerinckii mutant RT66 with considerable inhibitor-tolerance obtained by continuous culture was used for butanol production from non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). In fed-batch fermentation, 1.8L of diluted SAHHC containing 10 g/L of reducing sugar was provided during the acidogenic phase and 0.2L of concentrated SAHHC containing 300 g/L of reducing sugar was provided during the solventogenic phase. The mutant produced a total amount of solvents of 12.9 g/L, which consisted of 3.1 g/L of acetone, 9.3 g/L of butanol and 0.5 g/L of ethanol. A solvent yield of 0.35 g/g sugar and a productivity of 0.18 g/L h in 72 h were achieved. The remarkable inhibitor-tolerance of C. beijerinckii RT66 demonstrates that this may be an excellent strain for butanol production from ligocellulosic materials.

(4R*,5R*)-Diethyl 2-(4-nitro-phen-yl)-1,3-dioxolane-4,5-dicarboxyl-ate.

In the title compound, C(15)H(17)NO(8), the nitro group is essentially coplanar with the aromatic ring [dihedral angle = 6.4 (3) Å]. The five-membered ring has a twist conformation. In the crystal, C-H⋯O inter-actions link the mol-ecules into a helical chain propagating along [010].

(4R*,5R*)-2-(4-Meth-oxy-phen-yl)-1,3-dioxolane-4,5-dicarboxamide.

In the title compound, C(12)H(14)N(2)O(5), the five-membered 1,3-dioxolane ring has a twisted conformation. In the crystal, N-H⋯O and C-H⋯O hydrogen bonds link the mol-ecules into a two-dimensional network lying parallel to the ab plane. There are also C-H⋯π inter-actions present in the crystal structure.

Clostridium beijerinckii mutant with high inhibitor tolerance obtained by low-energy ion implantation.

Clostridium beijerinckii mutant strain IB4, which has a high level of inhibitor tolerance, was screened by low-energy ion implantation and used for butanol fermentation from a non-detoxified hemicellulosic hydrolysate of corn fiber treated with dilute sulfuric acid (SAHHC). Evaluation of toxicity showed C. beijerinckii IB4 had a higher level of tolerance than parent strain C. beijerinckii NCIMB 8052 for five out of six phenolic compounds tested (the exception was vanillin). Using glucose as carbon source, C. beijerinckii IB4 produced 9.1 g l(-1) of butanol with an acetone/butanol/ethanol (ABE) yield of 0.41 g g(-1). When non-detoxified SAHHC was used as carbon source, C. beijerinckii NCIMB 8052 grew well but ABE production was inhibited. By contrast, C. beijerinckii IB4 produced 9.5 g l(-1) of ABE with a yield of 0.34 g g(-1), including 2.2 g l(-1) acetone, 6.8 g l(-1) butanol, and 0.5 g l(-1) ethanol. The remarkable fermentation and inhibitor tolerance of C. beijerinckii IB4 appears promising for ABE production from lignocellulosic materials.

Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields.

With 30 g glucose/l as carbon source, Clostridium beijerinckii ART124, a mutant created by atmospheric pressure glow discharge, produced 13.7 g total solvent/l (containing 3.1 g acetone/l, 10.4 g butanol/l and 0.2 g ethanol/l) in 72 h. The mutant could also use sucrose or xylose or a mixture of glucose/xylose/arabinose with nearly equal yields.

Influence of osmotic stress on fermentative production of succinic acid by Actinobacillus succinogenes.

This study investigated the influence of osmotic stress on succinic acid production by Actinobacillus succinogenes NJ113. Both cell growth and succinic acid production were inhibited with the increase in osmotic stress of the medium. The use of three different osmoprotectants in the production of succinic acid was studied in order to decrease the inhibitory effects of osmotic stress during fermentation. Results indicated that proline offers optimal osmoprotection in the production of succinic acid by A. succinogenes NJ113. In tests of batch fermentation, the maximum cell concentration was observed to be 5.36 g DCW/L after the addition of 25 mmol/L proline to the fermentation medium. The cell concentration was 24% higher than that noted for the control. A total quantity of 56.2 g/L of succinic acid was produced, with a production rate of 1 g/L per hour, after 56 h of fermentation. The concentration and productivity of succinic acid was observed to be increased by 22.2% and 22%, respectively, as compared with the control. The specific activity levels of key enzymes in the metabolic network was noted to be higher following the addition of proline, particularly in the later stages of fermentation. This method of enhancing succinic acid production by the addition of an osmoprotectant may potentially provide an alternative approach for enhanced production of other organic acids.